Gastroenterology

Gastroenterology

Volume 158, Issue 4, March 2020, Pages 1029-1043.e10
Gastroenterology

Original Research
Full Report: Basic and Translational—Liver
Oncogenic Mutations in Armadillo Repeats 5 and 6 of β-Catenin Reduce Binding to APC, Increasing Signaling and Transcription of Target Genes

https://doi.org/10.1053/j.gastro.2019.11.302Get rights and content

Background & Aims

The β-catenin signaling pathway is one of the most commonly deregulated pathways in cancer cells. Amino acid substitutions within armadillo repeats 5 and 6 (K335, W383, and N387) of β-catenin are found in several tumor types, including liver tumors. We investigated the mechanisms by which these substitutions increase signaling and the effects on liver carcinogenesis in mice.

Methods

Plasmids encoding tagged full-length β-catenin (CTNNB1) or β-catenin with the K335I or N387K substitutions, along with MET, were injected into tails of FVB/N mice. Tumor growth was monitored, and livers were collected and analyzed by histology, immunohistochemistry, and quantitative reverse-transcription polymerase chain reaction. Tagged full-length and mutant forms of β-catenin were expressed in HEK293, HCT116, and SNU449 cells, which were analyzed by immunoblots and immunoprecipitation. A panel of β-catenin variants and cell lines with knock-in mutations were analyzed for differences in N-terminal phosphorylation, half-life, and association with other proteins in the signaling pathway.

Results

Mice injected with plasmids encoding K335I or N387K β-catenin and MET developed larger, more advanced tumors than mice injected with plasmids encoding WT β-catenin and MET. K335I and N387K β-catenin bound APC with lower affinity than WT β-catenin but still interacted with scaffold protein AXIN1 and in the nucleus with TCF7L2. This interaction resulted in increased transcription of genes regulated by β-catenin. Studies of protein structures supported the observed changes in relative binding affinities.

Conclusion

Expression of β-catenin with mutations in armadillo repeats 5 and 6, along with MET, promotes formation of liver tumors in mice. In contrast to N-terminal mutations in β-catenin that directly impair its phosphorylation by GSK3 or binding to BTRC, the K335I or N387K substitutions increase signaling via reduced binding to APC. However, these mutant forms of β-catenin still interact with the TCF family of transcription factors in the nucleus. These findings show how these amino acid substitutions increase β-catenin signaling in cancer cells.

Section snippets

Catalogue of Somatic Mutations in Cancer Database Analysis

To obtain the cancer-related CTNNB1 mutations depicted in Figure 1, we analyzed the COSMIC Web site (https://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=CTNNB1), filtering it on amino acids 310–440. Data were updated until January 2019.

Plasmids and Construction

N-terminal FLAG-tagged β-catenin variants were constructed by using the pcDNA 5′ UT-FLAG vector, as previously described.13 Briefly, the constructs of WT (WT), S33Y, and exon 3 deletion were generated by using the Gibson assembly method New England Biolabs (NEB).

K335I and N387K β-Catenin Variants Are Potent Inducers of Hepatocellular Carcinoma Formation in Mice

To directly demonstrate the oncogenic potential of the β-catenin K335I and N387K variants, we hydrodynamically injected them in the tail vein of mice.21 WT β-catenin was taken along as control. All variants were cotransfected with c-Met because previous work showed that oncogenic β-catenin alone is insufficient for hepatocellular carcinoma (HCC) development.16,17 In strong contrast to the mice injected with WT β-catenin, all K335I/N387K injected mice became moribund 6–7 weeks after injection,

Discussion

The β-catenin signaling pathway is one of the most commonly deregulated pathways among cancers.1 In colorectal cancers, this is predominantly accomplished by inactivating APC mutations, whereas in liver cancers, for example, aberrant activation has been mainly attributed to activating mutations in the CTNNB1 gene (20%–25%).4,7,33, 34, 35 The exon 3–related β-catenin mutations acquire enhanced signaling activities by interfering with proper N-terminal phosphorylation and subsequent proteolytic

Acknowledgments

Author contributions: Pengyu Liu performed the majority of experimental work and data analysis and authored the manuscript; Binyong Liang, Manning Qian, and Xin Chen performed and coordinated the animal experiments involving hydrodynamic transfection; Menggang Liu, Marla Lavrijsen, and Shan Li assisted with the experiments; Joyce H.G. Lebbink performed all the structural protein analyses and authored parts of the manuscript; Maikel P. Peppelenbosch supervised the project and improved the

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    Conflicts of interest The authors disclose no conflicts.

    Funding This research was financially supported by a China Scholarship Council PhD fellowship to Pengyu Liu (file no. 2014 0822 0029), Shan Li (file no. 2014 0806 0053) and Manning Qian (file no. 2018 0832 0464) and a National Institutes of Health grant (R01CA204586) to Xin Chen. Joyce Lebbink is supported by the gravitation program (024.001.028) from The Netherlands Organization for Scientific Research (NWO).

    Author names in bold designate shared co-first authorship.

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